Molecular Mechanism Of Wx Alleles’ Expression In Response To High Temperature And Its Breeding Design

With the intensification of the greenhouse effect,climate warming has become an environmental problem concerned all over the world.The frequent extreme high temperature in summer not only affects the yield of rice but also leads to the decrease of rice quality.Although the current level of rice production can solve people’s eating problems,with the improvement of living standards,people tend to choose high-quality rice with good taste,easy to chew and easy to absorb,which puts forward higher requirements for high-quality rice breeding under the condition of global warming.Starch is the main component of rice endosperm,and its content and composition are the key factors to determine the quality of rice.Among them,amylose content controlled by Wx is the top priority to determine rice cooking and eating quality,and is an important entry point for rice quality improvement research.Wx expression is regulated in a very complex way.However,relevant studies are not systematic and need further investigation.In this study,three typical Wx alleles(Wxlv,Wxa and Wxb)were selected as the research object,and the near-isogenic lines,site-specific mutations and transgenic rice materials with different promoters were used for high temperature treatment at the grain-filling stage.The comparison of the expression levels of three Wx alleles in response to high temperature and their effects on amylose contents were systematically analyzed,and the internal molecular mechanisms of these alleles were explored in terms of transcriptional initiation,post-transcriptional splicing and protein characteristics.Based on the above work,the key SNP sites that determine GBSSI protein stability and amylose synthesis in response to high temperature were identified,and transgenic function verification and molecular breeding design were carried out to create new rice materials with high temperature tolerance of starch structure and good seed appearance quality.Alternative splicing is an important mode of gene expression regulation in plant growth and development and environment responses.The splicing characteristic of Wx is the main reason for the difference of amylose content between Wxa and Wxb.However,the underlying molecular mechanism has not been reported.In this study,the splicing patterns of starch synthesis related genes including Wx were analyzed under high temperature.After identifying the alternative splicing sites of Wx,RNA binding proteins of the alternative splicing sites were screened.The splicing inhibitor RBP21,which specifically binds to the precursor mRNA of Wxb,was successfully identified by transgenic functional analysis.It is the first time to reveal the root cause of the difference of splicing efficiency of the first intron between Wxa and Wxb.In addition,the appearance quality of rbp21 mutant grains was better than that of wild type at high temperature.Therefore,gene editing of RBP21 is expected to create new rice materials with fine-tuning amylose content and better appearance quality under high temperature.The main research results are as follows:1.Study on the molecular mechanism of different Wx alleles in response to high temperature and breeding design:compared with Wxb,amylose contents controlled by Wxlv and Wxa were much higher;Compared with Wxlv and Wxb,the expression level of Wxa and it controlled amylose content were more stable under high temperature.The promoter activity of Wx and the splicing efficiency of mature mRNA were the decisive factors leading to the difference in the expression of different Wx alleles and their controlled amylose contents.SNP(Ex115C/T)resulted in the differences of the alternative splicing in Wx and the protein stability of GBSSI under high temperature,which further determined the high temperature tolerance of amylose synthesis in rice materials containing different Wx alleles.CRISPR/Cas9 was used to edit the single base of the SNP(Ex10-115C to T)in NPB and Nangeng 46,and it was found that the amylose contents in these rice materials with single base mutation was basically unaffected by high temperature and the seed appearance quality was much better than those of wild type,indicating that the SNP site could be applied to the breeding improvement of highquality rice with high temperature tolerance.2.Screening and functional identification of Wx splicing regulators:In this study,3rdgeneration full-length transcriptome sequencing was performed on rice seeds under high temperature,focusing on the analysis of the alternative splicing patterns of starch synthesis related genes including Wx and their response to high temperature.On the basis of identifying the splicing site of Wx,the RNA binding protein RBP21,which specifically binds with the splicing site in the first intron of Wxb,was screened and verified by RNA-pull down/MS and EMSA analysis,and the loss-function-mutants were created by CRISPR/Cas9 system.The splicing efficiency of the first intron of Wxb and amylose content in mature seeds were significantly up-regulated in rbp21 mutants.The appearance quality of rbp21 mutant seeds was better than that of wild type under high temperature.In addition,gene editing was carried out for RBP21 in other rice varieties such as Nangeng 46,which is expected to obtain new rice materials with fine-tuning amylose content and better appearance at high temperature.